White bands in western blotting - (Apr/12/2015 )

Hi

I am trying to do IP to pull down my protein of interest (80kd).  I am frequently to see white bands in lanes for whole cell lysate, pre-clear, post IP (IgG and my protein).

But I can still see my dark bands from my pull down.  What is the problem here?

The antibody concentration is not the problem since we used to do that.

Raku

I'm not quite sure what you mean by "white bands". Do you mean that the specific bands show up as white in the samples you mentioned?

If so, this is the result of there being "too much" protein present and as you then have a lot of antibody binding to it, it uses up the (presumably) HRP substrate faster than it can diffuse/ move into the area.

Hi thx for the reply but I was using licor instead of ECL.

it's possible that the bands are more concentrated than the blocking agent. that could reduce background where the bands are situated making them appear white.

It could be too much of antibody or protein concentration.